The widespread bacterial phosphoenolpyruvate (PEP) sugar phosphotransferase system is capable of carrying out the coupled translocation and phosphorylation of numerous sugars. The system is composed of two cytoplasmic proteins (Enzyme-I and HPr) that are used for all sugars and sugar-specific proteins known as Enzyme-II. We are currently performing structure/function studies on Enzyme-I (MW=63,500) from E. coli. Various point mutants of Enzyme-I at position 338 in the C-terminal domain have been cloned and have different enzyme activities. Most of 338-mutant proteins cannot be phosphorylated by PEP but can participate in a reversible phosphotransfer with HPr. Differential scanning calorimetry (DSC) shows that some of the thermal profiles of the mutant proteins are different from that of the wild type Enzyme-I. The N-terminal domain of Enzyme-I (EI-N, MW=28,700) has been cloned and also can participate in a reversible phosphotransfer with HPr but cannot be phosphorylated by PEP. The DSC profile of EI-N shows two endotherms at 54 and 59 degrees C in a first calorimetric upscan and an interesting thermal conversion on subsequent reheating cycles. The thermal conversion involves a decrease in area of the lower temperature endotherm and an inrease in the area of the 59 degrees C-endotherm while the total area remains unchanged. However, the overall secondary structure (as measured by circular dichroism spectra in the far-UV) remains essentially the same during reheating cycles. The thermal profiles of the mutants that cannot be phosphorylated by PEP but can accept phosphate from phosphorylated HPr are more similar to the thermal profile of EI-N than are the 338-mutants that have both functions.